WO2019160150A1 - Novel compound based on valerolactone, and medicine - Google Patents

Abstract

This novel valerolactone-based compound is represented by formula (I). [In the formula, X represents an allyl group, an aryl group, an ethynyl group, or a butenyl group. Y represents a single bond or a hydroxymethylene group, and Z represents oxygen, a methylene group, or an imide group. In the formula, Y represents a single bond or a hydroxymethylene group, m is 0 or 1, and n is 1 or 2.]

Description

Valerolactone new compound and medicine

The present invention relates to a novel valerolactone compound and a medicine. Specifically, the present invention relates to a neurodegenerative disease including amyotrophic lateral sclerosis (ALS) and a drug effective for the treatment of stroke.

In amyotrophic lateral sclerosis (ALS), an intractable disease, the motor nerves at the upper and lower levels of the corticospinal tract (pyramidal tract) that control voluntary movement are eventually degenerated. Respiratory muscles are paralyzed and fall into a state of inability to communicate under artificial respiration. Currently there are only two drugs for this disease. Riluzole (2-amino-6- (trifluoromethoxy) benzothiazole), a glutamate neurotransmission inhibitor, has a slight (2 to 3 months) life-prolonging effect on ALS (Non-patent Document 1). In addition, edaravone (edaravone; 3-methyl-1-phenyl-2-pyrazolin-5-one), a radical scavenger, has recently been additionally approved as an agent for inhibiting the progression of dysfunction in ALS.

There are approximately 1.18 million stroke patients in Japan, and 130,000 deaths from cerebral infarction annually. Cerebral infarction (ischemic stroke), which accounts for 80% of strokes, is caused by thrombotic occlusion of deep cortical arterioles (lacuna infarction), embolism due to cardiogenic embolism, and arterial thrombosis with decreased cerebral blood flow Based on this, sudden consciousness disturbance and neurological dysfunction (unilateral sensory loss and motor paralysis) occur. The direct cause of stroke is a blood clot based on blood coagulation, but symptoms are greatest in the acute phase (onset in a few minutes) in infarcted stroke, and brain tissue damage takes 24 to 48 hours in some progressive strokes Progresses step by step. The irreversible brain dysfunction, that is, neuronal cell death, that occurs in this advanced stage involves excessive release of glutamic acid, an excitatory amino acid, and oxidative stress associated therewith. In fact, the cerebrospinal fluid of patients with progressive stroke has a significantly higher glutamate concentration than normal (Non-patent Document 2). For treatment of seizures, it is best to apply t-PA (plasminogen activator) or the like within 4.5 hours of seizures. However, when reperfusion is impossible, in the penumbra, which is the peripheral region of the ischemic site, excessive Ca ²⁺ influx into the neuron via the NMDA glutamate receptor occurs as time elapses from the attack. At the same time, the reactive oxygen species rises, and in combination with the inflammatory response, apoptosis and necrosis of neurons occur, and the site falls into irreversible dysfunction (Non-patent Document 3). Although edaravone can suppress this process by the action of scavenging free radicals, the time for which this drug can be applied is limited to 24 hours after the attack. On the other hand, NMDA-type glutamate receptor antagonists that are effective in experimental treatments have strong side effects, and no drug is applied (Non-patent Document 4). Only antiplatelet therapy (such as argatroban) is used as a conservative therapy.

So far, new low molecular weight ALS therapeutic candidates based on pathological research include (1) motor neuroprotection (survival maintenance) action, (2) antioxidant action, (3) antiglutamate neurotoxicity, (4) anti-inflammatory A group of molecules focused on the effects of the above showed effectiveness in experimental treatment of ALS model animals. However, among those small molecule ALS therapeutic candidates, coenzyme Q10, N-acetylcysteine, methicobalamin, and glutathione have not shown efficacy in clinical trials.

In addition, a considerable number of trials for administering a protein having a neuroprotective action or introducing the protein-encoding gene with a viral vector have progressed to clinical trials. Among them, insulin-like growth factor-1 (IGF-1) was expected to have an effect of preventing motor nerve loss, but the clinical trial was unsuccessful in the third phase (Non-patent Document 5).

3rd ALS treatment strategy includes cell transplantation. In addition to fetal motor nerves, attempts have been made to transplant glial cells capable of supplying neuroprotective factors into the spinal cord. Furthermore, transplantation of iPS cells, mesenchymal stem cells, etc. with little rejection reaction has been attempted, but so far, the effectiveness of symptom improvement and the like has not been shown.

As described above, there are currently only two drugs approved as ALS treatment drugs, and it cannot be said that the ALS treatment effect is high. Therefore, development of a new ALS therapeutic drug is desired. In addition, since ALS is considered to be associated with a plurality of factors, development of a drug having an action mechanism different from that of existing drugs is required. In addition, as described above, there is no therapeutic drug for ischemic stroke that can be used in the subacute phase, and there is a demand for the development of a drug that can be applied to ischemic stroke in the subacute phase.

The present invention has been made in view of the above circumstances, and an object thereof is to provide a novel compound used as a medicament for treating or preventing neurodegenerative diseases including ALS. Moreover, it aims at providing the novel compound utilized as a pharmaceutical which can be used as a novel inhibitor of the progression of cerebral infarction in the advanced stage.

The present invention is characterized by being a novel compound of the valerolactone type represented by the formula (I).

[Wherein X is an allyl group, an aryl group, an ethynyl group, or a butenyl group. Y is a single bond or a hydroxymethylene group, and Z is an oxygen, methylene group or imide group. m is 0 or 1, and n is 1 or 2. ]

The compound represented by the formula (I) is preferably represented by the following formula (I-1).
It is.

The present invention further provides a novel compound of valerolactone (Valerolactone) represented by the above formula as a main component, main component, active ingredient, or useful component, depending on the use, in a predetermined amount, for example, the use. It is a pharmaceutical or a pharmaceutical composition that is contained in a range of not less than the minimum content and not more than the limit content to achieve. The compound (I) is preferably used as a medicament for treating and / or preventing neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) or stroke. As described below, the inventor applied the compound (I) to a plurality of individuals in amounts of 1 mg / Kg, 5 mg / Kg, and 10 mg / Kg. In all cases, the efficacy was confirmed. Therefore, the effective dose may be selected, for example, within the range of 1 mg / Kg to 10 mg / Kg. Furthermore, when the inventor confirmed the blood and main organs of a plurality of individuals to which the compound was continuously administered, no side effects or abnormalities corresponding to adverse events were observed. The medicament includes at least one of a compound represented by the above formula, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable solvate thereof, and a pharmaceutical agent. It may contain an acceptable carrier.

According to the present invention, a drug for treating or preventing a neurodegenerative disease including ALS, and a novel compound used as an active ingredient of the drug are provided. Furthermore, a medicament that can be used as a novel inhibitor of progression of cerebral infarction at an advanced stage, and the medicament are provided.

Pleosporales sp. The isolation flow of compound (I-1) and compound (I-2) from the culture solution of NUH322 strain is shown. Pleosporales sp. The isolation flow of compound (I-3) and compound (I-4) from the culture solution of NUH322 strain is shown. 1 shows the correlation between ¹ H- ¹ H COSY and HMBC of Compound (I-1). 1 shows the NOESY correlation of compound (I-1). Dif. Of compound (I-1). The result of NOE is shown. ¹ shows the correlation between ¹ H- ¹ H COSY and HMBC of compound (I-2). 1 shows the NOESY correlation of compound (I-2). Compounds showing a correlation (I-3) of ¹ H- ¹ H COZY and HMBC. 1 shows the NOESY correlation of compound (I-3). The result of the ROS assay of ethyl acetate crude extract (322-Ext) and compound (I-1) (YY-1) in the culture solution of NUH322 strain is shown. The vertical axis shows the relative intensity with respect to 30 μg / mL (120 μM) trolox (positive control). For the concentration of the test substance performed 3 times or more, the standard error is obtained, and the result is expressed as an average value ± standard error. The result of the ODAP-EA assay of ethyl acetate crude extract (322-Ext) and compound (I-1) (YY-1) of NUH322 strain culture solution is shown. The vertical axis shows the relative intensity with respect to 4 mM N-acetylcysteine (NAC; positive control). For the concentration of the test substance performed 3 times or more, the standard error is obtained, and the result is expressed as an average value ± standard error. The result of the Dual-luciferase assay of ethyl acetate crude extract (322-Ext) and compound (I-1) (YY-1) in the culture solution of NUH322 strain is shown. The vertical axis shows the relative intensity with respect to the solvent (DMSO). The standard error was calculated | required about the density | concentration of the test substance performed 3 times or more, and the result was shown by the average value +/- standard error. The upper figure shows the luminescence intensity of firefly luciferase, and the lower figure shows the luminescence intensity of Renilla luciferase. The transition of the body weight of the mouse | mouth in the administration test 1 of Example 3 is shown. The mean value ± standard error of the body weight of each group of mice up to about 125 days after birth was shown. The vertical axis represents the average weight of mice, and the horizontal axis represents the number of days after birth. “YY-1 (1)” indicates a YY-1 (1 mg / kg) administration group, and “YY-1 (10)” indicates a YY-1 (10 mg / kg) administration group. The result of having analyzed the survival days of the mouse | mouth in the administration test 1 of Example 3 by Kaplan-Meier method is shown. It is an analysis result of a YY-1 (10 mg / kg) administration group. The result of having analyzed the survival days of the mouse | mouth in the administration test 1 of Example 3 by Kaplan-Meier method is shown. It is an analysis result of a YY-1 (1 mg / kg) administration group. The result of having analyzed the number of days until the onset of the movement disorder of the mouse | mouth in the administration test 2 of Example 3 by Kaplan-Meier method is shown. The result of having analyzed the survival days of the mouse | mouth in the administration test 2 of Example 3 by Kaplan-Meier method is shown.

[Pharmaceutical composition]
In one embodiment, the present invention comprises a compound represented by the following general formula (I), a stereoisomer thereof, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable solvate thereof. Provided is a medicament or a pharmaceutical composition comprising at least one compound selected from the group and a pharmaceutically acceptable carrier.

[Wherein X is an allyl group, an aryl group, an ethynyl group, or a butenyl group. Y is a single bond or a hydroxymethylene group, and Z is an oxygen, methylene group or imide group. m is 0 or 1, and n is 1 or 2. ]

The compound represented by the general formula (I) may have a stereoisomer. General formula (I) may represent these stereoisomers as a representative. Hereinafter, the compound represented by the general formula (I) and the stereoisomers thereof are also referred to as “compound (I)”. As compound (I), those stereoisomers may be used alone or a mixture of these stereoisomers may be used. “Stereoisomers” include enantiomers and diastereomers.

Specific examples of compound (I) include the following compounds (I-1) to (I-4).

Compound (I) may be in the form of a pharmaceutically acceptable salt. “Pharmaceutically acceptable salt” means a salt that does not inhibit the pharmacological action (ROS elimination action, excitotoxicity reduction action, SOD1-G93A genotoxicity reduction action, etc.) of compound (I). The pharmaceutically acceptable salt is not particularly limited, and for example, a salt with an alkali metal (sodium, potassium, etc.); a salt with an alkaline earth metal (magnesium, calcium, etc.); an organic base (pyridine, triethylamine, etc.) Salt with amine, salt with amine, salt with organic acid (acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, etc.) And salts with inorganic acids (hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, nitric acid, etc.). Preferred examples of the pharmaceutically acceptable salt of compound (I) include salts with alkali metals.

Compound (I) may be in the form of a pharmaceutically acceptable solvate. A “solvate” is a complex formed by the compound (I) with a solvent. "Pharmaceutically acceptable solvate" means a solvate that does not inhibit the pharmacological action of compound (I). The solvate of compound (I) is not particularly limited, and examples thereof include hydrates and ethanol solvates. Compound (I) may be in the form of a solvate of a pharmaceutically acceptable salt.

The pharmaceutical composition of the present embodiment comprises compound (I), a pharmaceutically acceptable salt of compound (I), a pharmaceutically acceptable solvate of compound (I), and a pharmaceutical of compound (I). It may contain a mixture of two or more selected from the group consisting of solvates of acceptable salts. Hereinafter, compound (I), pharmaceutically acceptable salt of compound (I), pharmaceutically acceptable solvate of compound (I), and solvent of pharmaceutically acceptable salt of compound (I) Japanese products may be collectively referred to as “compound (I) etc.”.

The applicable disease of the pharmaceutical composition of the present embodiment is not particularly limited, and preferred examples include neurodegenerative diseases and stroke. Therefore, a pharmaceutical composition for treating or preventing a neurodegenerative disease or stroke containing Compound (I) and the like is a suitable example of the pharmaceutical composition of the present embodiment. “Neurodegenerative disease” refers to a disease in which progressive neuronal cell death occurs. Examples of neurodegenerative diseases include ALS, Alzheimer's disease, Parkinson's disease, Huntington's disease, multisystem atrophy, spinocerebellar degeneration, spinal and spinal muscular atrophy, spinal muscular atrophy, and primary lateral sclerosis. However, it is not limited to these. “Stroke” refers to a disease in which blood circulation disorders occur in the blood vessels of the brain. Stroke includes, but is not limited to, cerebral infarction resulting from thrombosis or infarction.

As shown in Examples described later, Compound (I) is composed of (1) reactive oxygen species (ROS elimination ability), (2) excitatory cell death of motor nerve via glutamate receptor (Excitotoxicity) It is a compound that has been confirmed to have a suppressive action and (3) an adverse action due to the appearance of abnormal protein (Proteinopathy) due to the introduction of the SOD1-G93A gene. Furthermore, in an in vivo test using ALS model mice, there was a tendency to prolong survival and development of movement disorders. Therefore, compound (I) can be suitably used for neurodegenerative diseases related to the above (1) to (3). ALS etc. are illustrated as such a neurodegenerative disease. Especially, the pharmaceutical composition of this embodiment can be used suitably in order to treat or prevent ALS. In addition, in the stroke, neuronal apoptosis and necrosis occur in the subacute phase due to an inflammatory reaction due to an increase in ROS and excessive Ca ²⁺ influx into the neuron via the NMDA type glutamate receptor. Since the compound (I) has the actions (1) and (2), it can be suitably used for treating stroke (particularly stroke in the subacute stage).

In addition, compound (1) is characterized in that among the activities (1) to (3) described above, (2) motor nerve excitatory cell death inhibitory action is high. Therefore, for example, the pharmaceutical composition of the present embodiment may be applied to a subject (such as a patient suffering from a neurodegenerative disease) for which no effect was observed with edaravone, which is a radical scavenger.

The application target of the pharmaceutical composition of the present embodiment is preferably an animal that develops a neurodegenerative disease. For example, the pharmaceutical composition of the present embodiment can be suitably used for humans or mammals other than humans. Mammals other than humans are not particularly limited, but include primates (monkeys, chimpanzees, gorillas, etc.), rodents (mouses, hamsters, rats, etc.), rabbits, dogs, cats, cows, goats, sheep, horses, etc. It is done.

The pharmaceutical composition of the present embodiment may contain at least one pharmaceutically acceptable carrier in addition to compound (I) and the like. "Pharmaceutically acceptable carrier" means a carrier that does not inhibit the physiological activity of the active ingredient and does not exhibit substantial toxicity to the administration subject. By “not exhibiting substantial toxicity” is meant that the ingredient is not toxic to the administered subject at the doses normally used. Pharmaceutically acceptable carriers include any known pharmaceutically acceptable ingredients that are typically considered inactive ingredients. The pharmaceutically acceptable carrier is not particularly limited, and examples thereof include solvents, diluents, vehicles, excipients, glidants, binders, granulating agents, dispersing agents, suspending agents, wetting agents, Lubricants, disintegrants, solubilizers, stabilizers, emulsifiers, fillers, preservatives (for example, antioxidants), chelating agents, flavoring agents, sweeteners, thickeners, buffers, colorants, etc. Can be mentioned. A pharmaceutically acceptable carrier may be used alone or in combination of two or more.

The pharmaceutical composition of the present embodiment may contain other components. Other components are not particularly limited, and those commonly used in the pharmaceutical field can be used without particular limitation. Moreover, this pharmaceutical composition may contain active ingredients other than compound (I). Active ingredients include, for example, vitamins and derivatives thereof, anti-inflammatory agents, anti-inflammatory agents, blood circulation promoters, stimulants, hormones, stimulant mitigants, analgesics, cell activators, plant / animal / microbe extracts, antipruritic agents Agents, anti-inflammatory analgesics, antifungal agents, antihistamines, hypnotic sedatives, tranquilizers, antihypertensives, antihypertensive diuretics, antibiotics, anesthetics, antibacterial substances, antiepileptics, coronary vasodilators, herbal medicines Examples include, but are not limited to, glazes and keratin softening release agents. Other components may be used alone or in combination of two or more.

The dosage form of the pharmaceutical composition of the present embodiment is not particularly limited, and can be a dosage form generally used as a pharmaceutical preparation. The pharmaceutical composition of the present embodiment may be an oral formulation or a parenteral formulation. Examples of oral preparations include tablets, coated tablets, pills, powders, granules, capsules, syrups, fine granules, liquids, drop love, and emulsions. Examples of parenteral preparations include injections, suppositories, ointments, sprays, external preparations, ear drops, eye drops, nasal drops, inhalants and the like. The pharmaceutical composition of these dosage forms can be formulated according to a conventional method (for example, a method described in the Japanese Pharmacopoeia).

The administration route of the pharmaceutical composition of the present embodiment is not particularly limited, and can be administered by an oral or parenteral route. The parenteral route includes all routes other than oral administration such as intravenous, intramuscular, subcutaneous, intranasal, intradermal, ophthalmic, intracerebral, intrarectal, intravaginal, intraperitoneal, etc. To do. Administration may be local administration or systemic administration.

In the pharmaceutical composition of the present embodiment, a therapeutically effective amount of compound (I) can be administered. “Therapeutically effective amount” means an amount of a drug effective for treating or preventing a target disease. For example, when the pharmaceutical composition of the present embodiment is for treating or preventing a neurodegenerative disease, the therapeutically effective amount of compound (I) or the like is an amount capable of delaying the progression of the neurodegenerative disease. obtain. The therapeutically effective amount may be appropriately determined according to the patient's symptoms, body weight, age, sex, etc., the dosage form of the pharmaceutical composition, the administration method, and the like. For example, the pharmaceutical composition of the present embodiment can be 0.01 to 500 mg per kg body weight of the administration subject as a single dose of compound (I) and the like. The dosage may be 0.15-500 mg / kg, 0.5-300 mg / kg, 1-200 mg / kg, 1-100 mg / kg. Also good.

The pharmaceutical composition of the present embodiment may contain a therapeutically effective amount of compound (I) and the like per unit dosage form. For example, the content of compound (I) and the like in the pharmaceutical composition of the present embodiment may be 0.01 to 80% by mass, 0.05 to 50% by mass, 0.1 to It may be 30% by mass.

The administration interval of the pharmaceutical composition of the present embodiment may be appropriately determined depending on the patient's symptoms, weight, age, sex, etc., the dosage form of the pharmaceutical composition, the administration method, and the like. The administration interval can be, for example, once every several hours, once a day, once every 2-3 days, or the like.

The pharmaceutical composition of the present embodiment may be used in combination with other pharmaceuticals. For example, it can be used in combination with other therapeutic agents for neurodegenerative diseases. For example, when the pharmaceutical composition of the present embodiment is applied to ALS, it may be used in combination with riluzole, edaravone and the like.

<Method for Producing Compound (I)>
Compound (I) is a fungus belonging to the order of Pleosporales, Pleosporales sp. It is a compound isolated from the culture solution of NUH322 strain (hereinafter also referred to as “NUH322 strain”). Therefore, it can be produced from the culture solution of this strain by performing isolation / purification by combining known isolation / purification techniques. Alternatively, it can be produced by chemical synthesis by combining known chemical reactions.

(Method using culture medium of NUH322 strain)
Compound (I) was prepared by using Pleosporales sp. It is a compound isolated from the culture solution of NUH322 strain (hereinafter also referred to as “NUH322 strain”), and can be isolated and purified from the culture solution of NUH322 strain. The NUH322 strain is a filamentous fungus isolated from red pine deciduous leaves of the Sugadaira Plateau, Ueda City, Nagano Prefecture, Japan. The NUH322 strain was issued on January 31, 2018, under the accession number: NITE P-02624, which was established by the National Institute of Technology and Evaluation of Microorganisms (2-5-8, Kazusa Kamashika, Kisarazu City, Chiba Prefecture, Japan). It is deposited domestically. On February 13, 2019, the center received a transfer request to the international deposit of the NUH322 stock that was domestically deposited under the receipt number: NITE ABP-02624. The request for transfer related to this receipt number will be issued with a receipt under the Budapest Treaty concerning the international recognition of the deposit of microorganisms under the patent procedure. Specific examples of the method for isolating compound (I) from NUH322 strain are shown below.

First, liquid culture of NUH322 strain is performed. Liquid culture of the NUH322 strain can be performed using a liquid medium generally used for fungal culture. Examples of the liquid culture medium for NUH322 strain include the GP medium described in the Examples. For the culture, culture conditions generally used for fungal culture can be used. Examples of the culture conditions include shaking culture (eg, 150 rpm) at 20 to 30 ° C. (eg, 25 ° C.). The culture period is not particularly limited. For example, the culture may be performed until a steady state is reached. Examples of the culture period include 1 to 4 weeks or 2 to 3 weeks.

After culturing NUH322 strain, the culture solution is filtered using a Buchner funnel to separate the mycelium from the medium. The filtrate obtained by the filtration is extracted with ethyl acetate, and then compound (I) can be isolated by performing silica gel chromatography and high performance liquid chromatography (HPLC).

More specifically, the filtrate can be extracted with ethyl acetate by adding an equal amount of ethyl acetate to the filtrate and stirring at room temperature for about 1 to 5 hours. Subsequently, the ethyl acetate layer is collected and concentrated by a rotary evaporator or the like, whereby an ethyl acetate extract of the culture solution of NUH322 strain can be obtained.

Silica gel column chromatography can be performed by dissolving the ethyl acetate extract in an organic solvent such as methanol, loading it onto a silica gel column, and fractionating using an appropriate developing solvent. A commercially available filler can be used without particular limitation, and examples thereof include Wakogel (registered trademark) C-200 (Wako Pure Chemical Industries). As the developing solvent, for example, an ethyl acetate / hexane mixed solvent can be used.

HPLC can be performed using, for example, a mixed solvent of acetonitrile (CH ₃ CN) / water (H ₂ O) as a mobile solvent. A commercially available column for HPLC can be used without particular limitation, and examples thereof include Kaseisorb LC ODS-PH Super (Tokyo Kasei Kogyo).

For example, for the compound (I-1), a fraction of ethyl acetate / hexane = 1/1 was collected by silica gel column chromatography, and HPLC (mobile solvent: CH ₃ CN / H ₂ O = 25) of the fraction was collected. / 75) and collecting the fifth peak fraction.

For the compound (I-2), a fraction of ethyl acetate / hexane = 1/1 was collected by silica gel column chromatography, and HPLC (mobile solvent: CH ₃ CN / H ₂ O = 25/75) of the fraction was collected. And the third peak fraction is collected, and further, HPLC (CH ₃ CN / H ₂ O = 22/78) of the fraction is performed, and the first peak fraction is collected. be able to.

For the compound (I-3), a fraction of ethyl acetate / hexane = 1/0 was collected by silica gel column chromatography, and HPLC (mobile solvent: CH ₃ CN / H ₂ O = 18/82) of the fraction was collected. ) And collecting the seventh peak fraction.

For the compound (I-4), a fraction of ethyl acetate / hexane = 1/0 was collected by silica gel column chromatography, and HPLC (mobile solvent: CH ₃ CN / H ₂ O = 18/82) of the fraction was collected. ) And collecting the fourth peak fraction.

(Chemical synthesis method)
Compound (I) may be chemically synthesized by appropriately combining known chemical reactions.
For example, compound (I-4) can be prepared according to Nagarapu et al. (Tetrahedron 68 (2012) 5829-5832.). An example of a specific synthesis route is shown below. In the following chemical formula, “LiHMDS” represents hexamethyldisilazane lithium.

For example, the above-mentioned compound (I-1), compound (I-2) and compound (I-3) can be produced by the synthetic route shown below. In the following synthesis route, a synthesis method for compound (I-1) is representatively described. In the following chemical formula, “TBSCl” represents tert-butyldimethylchlorosilane. “TBAF” represents tetra-n-butylammonium fluoride.

[Compound]
In one embodiment, the present invention relates to a compound represented by the following general formula (I-1), a stereoisomer thereof, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable solvate thereof. A compound selected from the group consisting of:

Examples of the stereoisomer of compound (I-1) include the following compound (I-2) and compound (I-3).

Examples of the pharmaceutically acceptable salt of compound (I-1) or a stereoisomer thereof include the same as those exemplified in the above [Pharmaceutical composition]. Examples of the pharmaceutically acceptable solvate of compound (I-1) or a stereoisomer thereof include the same as those exemplified in the above [Pharmaceutical composition].

The compound (I-1) and stereoisomers thereof can be obtained by the method described in <Method for producing compound (I)> in the above [Pharmaceutical composition].

[Other Embodiments]
In one embodiment, the present invention relates to a compound represented by the above general formula (I), a pharmaceutically acceptable salt thereof, and their production in the manufacture of a pharmaceutical composition for treating or preventing a neurodegenerative disease. There is provided the use of at least one compound selected from the group consisting of pharmaceutically acceptable solvates.

In one embodiment, the present invention provides a compound represented by the above general formula (I), a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable salt thereof for use in treating or preventing a neurodegenerative disease. There is provided the use of at least one compound selected from the group consisting of:

In one embodiment, the present invention is at least selected from the group consisting of compounds represented by the above general formula (I), pharmaceutically acceptable salts thereof, and pharmaceutically acceptable solvates thereof. Provided is a method for treating a neurodegenerative disease, comprising administering one compound to a subject (eg, a patient suffering from a neurodegenerative disease, etc.).

In one embodiment, the present invention provides a compound represented by the above general formula (I), a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable salt thereof for treating or preventing a neurodegenerative disease. The use of at least one compound selected from the group consisting of solvates is provided.
In the above embodiment, the neurodegenerative disease is preferably ALS.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to the following examples.

[Example 1] Isolation of a compound A fungal extract library was prepared as a part of a natural product fraction-compound library owned by the Nihon University School of Pharmacy. Based on the results of ODAP-EA assay, Dual-luciferase assay, and ROS assay described below, Pleosporales sp. An extract of NUH322 strain (hereinafter referred to as “NUH322 strain”) was selected. The NUH322 strain is a fungus collected from red pine litter on the Sugadaira Plateau, Ueda City, Nagano Prefecture, Japan.

In a clean bench, the corn meal agar (CMA) plate medium was inoculated with the medium piece from the fungal stock, and then cultured at 25 ° C. for about 2 weeks until the mycelium was extended. After confirming that the mycelium was sufficiently extended on the plate medium, the plate medium was cut into a 2 mm grid, and about 15 pieces of each medium piece were inoculated into a glucose-peptone (GP) liquid medium. After inoculation, shaking culture was performed at 25 ° C. and 150 rpm for 3 weeks. After culture, the culture was filtered using a Buchner funnel to separate the mycelium and the medium. An equal amount of ethyl acetate was added to the filtered medium, and the mixture was stirred at room temperature with a magnetic stirrer for about 3 hours. Thereafter, the ethyl acetate layer was recovered and concentrated under reduced pressure using a rotary evaporator to obtain an ethyl acetate extract (1582.2 mg). Tables 1 and 2 show the compositions of the CMA plate medium and the GP liquid medium.

The ethyl acetate extract (1582.2 mg) was dissolved in methanol and fractionated by silica gel column chromatography (SiO ₂ CC: Wakogel® C-200). As developing solvents, ethyl acetate (EtOAc) / hexane and methanol (MeOH) were used to obtain fractions of EtOAc / hexane = 1/5, 1/1, 1/0 and MeOH fractions.

Subsequently, the fraction of the above EtOAc / hexane = 1/1 was fractionated by HPLC (Kaseisorb LC ODS-PH Super; Tokyo Chemical Industry). As the mobile solvent, CH ₃ CN / H ₂ O = 25/75 was used. Compound (I-1) was obtained from the fifth fraction separated by HPLC (209.5 mg).

The third peak separated by HPLC was collected and further fractionated by HPLC (Kaseisorb LC ODS-PH Super; Tokyo Chemical Industry). CH ₃ CN / H ₂ O = 22/78 was used as the mobile solvent. Compound (I-2) was obtained from the first fraction (1.8 mg).

The fraction of EtOAc / hexane = 1/0 was fractionated by HPLC (Kaseisorb LC ODS-PH Super; Tokyo Chemical Industry). As the mobile solvent, CH ₃ CN / H ₂ O = 18/82 was used. Compound (I-3) was obtained from the 7th fraction separated by HPLC (1.1 mg). In addition, compound (I-4) was obtained from the fourth fraction separated by HPLC (5.0 mg).

The isolation flow of the above compound is shown in FIG. 1 and FIG.

[Example 2] Structural analysis of compound <equipment used>
The following equipment was used for the structural analysis of the compounds.
NMR system: JEOL JNM-ECX500
Spectrometer using 3.0 mm microcells (Sigemi)
Mass spectrometer: Xevo G2-S QTof Quadrupole Time of Flight Mass Spectrometer (Waters)
Polarimeter: JASCO P-1020 Automatic Polarimeter (JASCO)
Infrared spectrophotometer: JASCO FT / IR-4100 FT-IR Spectrometer (JASCO)
UV-visible spectrophotometer: JASCO V-730BIO UV-VIS Spectrophotometer
Circular dichroism dispersometer: JASCO J-600 Circular Dichroism spectrometer

<Structural analysis of compound (I-1)>
Compound (I-1) is a pale yellow oily substance. From the results of HROFMS analysis, the molecular formula C ₁₀ H ₁₄ O ₃ [m / z 205.0841 [M + Na] ⁺ (calcd for C ₁₀ H ₁₄ O ₃ Na 205.0841, unsat 4)]. The specific rotation of compound (I-1) was [α] ¹⁷ _D -2.79 ° (c5.71 MeOH). Further, based on FT / IR measurement results, the presence of hydroxy groups from the absorption of 3422cm ^-1 is the presence of the carbonyl group from the absorption of 1765cm ^-1 is suggested. Furthermore, in the UV spectrum, the maximum was recognized at λ _max (MeOH) nm (log ε): 240.8 (5.38), 280.0 (5.82). ^{From the} results of spectral analysis of ¹ H-NMR, ¹³ C-NMR, DEPT (distortionless enhancement by polarization transfer), and HMQC (heteronuclear multiple quantum correlation), the presence of methyl group 1, methylene group 2, methine group 6, and quaternary carbon 1 Was suggested.

The ^{1 H- 1 H COSY (correlation spectroscopy} ), the correlation of the H-3 / H-4 / H-5 / H-6 / H-7 / H-8 / H-9 / H-10 / H 3 -11 Was observed, suggesting that C-3 to C-11 were bound. Since the chemical shift of H-6 was shifted to the low magnetic field side (δ _H 4.48), it was suggested that an O atom was bonded to C-6. Since a correlation of HMBC (heteronuclear multiple bond connectivity) to H-3 / C-2 and H-6 / C-2 was observed, compound (a1-1) has a six-membered ring structure containing a lactone. It was estimated that In addition, since the chemical shift of H-5 was shifted to the low magnetic field side (δ _H 4.52), it was suggested that C-5 has a hydroxy group. When ¹ H-NMR spectrum was measured by adding heavy water, the signal of δ _H 3.00 disappeared, so that it was estimated to be a hydroxy group signal.

The relative steric structure is dif. It estimated using NOE (difference nuclear overhauser effect), NOESY (nuclear overhauser effect spectroscopy), and coupling constant. The correlation by NOESY is seen in H-5 / H-7, H-7 / H-9, H-9 / H3-11, H-6 / H-8, H-8 / H-10, dif. Since OH-5 / H-6 was observed by NOE, the relative steric structure was estimated with H-5 in the α-position and H-6 in the β-position. The side chains C-7 to C-8 and C-9 to C-10 also have coupling constants of J _{H-7, H-8} = 15 Hz, J _{H-9, H-10} = 15 Hz, respectively. This estimate was supported.

¹ H- ¹ shows the correlation of H COZY and HMBC Figure 3A, shows the correlation of NOESY Figure 3B, dif. The NOE results are shown in FIG. 3C. Table 3 shows the measurement results (CDCl ₃ , 500 MHz) of ¹ H NMR and ¹³ C NMR.

From the above results, it was determined that the compound (I-1) had a relative steric structure represented by the following formula (I-1).

<Structural analysis of compound (I-2)>
Compound (I-2) is a pale yellow oily substance. From the result of HROFMS analysis, the molecular formula C ₁₀ H ₁₄ O ₃ [m / z 205.0839 [M + Na] ⁺ (calcd for C ₁₀ H ₁₄ O ₃ Na 205.0841, unsat 4)]. The specific rotation of the compound (I-2) was [α] ¹⁶ _D + 36.9 ° (c0.11, MeOH). Further, the presence of carbonyl group 2 was suggested based on the FT / IR measurement results {ν _max (KBr) 3447, 1638 cm ⁻¹ }. Furthermore, in the measurement of UV spectrum, the maximum was recognized by (lambda) _max (MeOH) nm (log (epsilon)): 240.8 (5.47), 295.2 (5.41). Spectral analysis results of ¹ H-NMR, ¹³ C-NMR, DEPT, and HMQC suggested the presence of methyl group 1, methylene group 2, methine group 6, and quaternary carbon 1.

^From the correlation between ¹ H- ¹ H COSY and HMBC, it was suggested that the planar structure of the compound (I-2) was the same as that of the compound (I-1).

Relative conformation was estimated using NOESY and coupling constants. Since the correlation of H-5 / H-9 and H-7 / H3-11 was observed by NOESY measurement, the relative steric structure represented by the following formula (I-2) was determined. The coupling constants of C-7 to C-8 and C-9 to C-10 are J _{H-7, H-8} = 11.0 Hz, J _{H-9, H-10} = 14.0 Hz. But this estimate was supported.

The correlation of ¹ H- ¹ H COSY and HMBC is shown in FIG. 4A, and the correlation of NOESY is shown in FIG. 4B. Table 4 shows the measurement results (CDCl ₃ , 500 MHz) of ¹ H NMR and ¹³ C NMR.

<Structural analysis of compound (I-3)>
Compound (I-3) is a pale yellow oily substance. From the result of HROFMS analysis, the molecular formula C ₁₀ H ₁₄ O ₃ [m / z 205.0841 [M + Na] ⁺ (calcd for C ₁₀ H ₁₄ O ₃ Na 205.0841, unsat 4)]. The specific rotation of compound (I-3) was [α] ¹⁷ _D −16.1 ° (c 0.09, MeOH). Further, based on FT / IR measurements, hydroxy group from the absorption of 3448cm ^-1 is the presence of the carbonyl group from the absorption of 1633 cm ^-1 was suggested. Furthermore, the measurement of the UV spectrum showed a _maximum at λ _max (MeOH) nm (log ε): 231.8 (6.56). Spectral analysis results of ¹ H-NMR, ¹³ C-NMR, DEPT, and HMQC suggested the presence of methyl group 1, methylene group 2, methine group 6, and quaternary carbon 1.

^From the correlation of ¹ H- ¹ H COSY and HMBC, it was suggested that the planar structure of compound (I-3) was the same as that of compound (I-1).

Relative conformation was estimated using NOESY and coupling constants. From the measurement of NOESY, the correlation of H-5 / H-7, H-9 / H ₃ -11 was observed, so the relative steric structure represented by the following formula (I-3) was estimated. The coupling constants of C-7 to C-8 and C-9 to C-10 are J _{H-7, H-8} = 6.0 Hz, J _{H-9, H-10} = 11.5 Hz. But this estimate was supported.

The correlation of ¹ H- ¹ H COSY and HMBC is shown in FIG. 5A, and the correlation of NOESY is shown in FIG. 5B. Table 5 shows the measurement results of ¹ H NMR and ¹³ C NMR (CD ₃ OD, 500 MHz).

<Structural analysis of compound (I-4)>
The measured values of ¹ H-NMR and ¹³ C-NMR of the compound (I-4) were in agreement with the literature values of Sapinofuranone A, a metabolite of the fungus Sphaeropsis sapnea (Evidente A et al., J Nat Prod. 1999 Feb; 62 (2): 253-6.). The measurement results of ¹ H-NMR and ¹³ C-NMR of the compound (I-4) are shown below.

¹ H-NMR (CD ₃ OD, 500 MHz) δ _H 2.37 (1H, dt, J = 16.0, 1.5 Hz, H-2), 2.49 (1H, m, H-2), 1.62 (1H, m, H -3), 1.87 (1H, m, H-3), 3.50 (1H, ddd, J = 9.5, 6.0, 3.0 Hz, H-4), 3.93 (1H, brt, J = 6.0 Hz, H-5) , 5.62 (1H, dd, J = 15.0, 7.0 Hz, H-6), 6.22 (1H, dd, J = 15.0, 10.5 Hz, H-7), 6.08 (1H, ddd, J = 15.0, 10.5, 1.5 Hz, H-8), 5.70 (1H, dq, J = 15.0, 7.0 Hz, H-9), 1.74 (3H, d, J = 7.0 Hz, H-10).

¹³ C-NMR (CD ₃ OD, 500 MHz) δ _C 178.0 (C-1), 31.7 (C-2), 29.0 (C-3), 75.0 (C-4), 76.8 (C-5), 130.8 (C-6), 133.6 (C-7), 132.5 (C-8), 130.3 (C-9), 18.2 (C-10).

Compound (I-4) was determined to have the same structure as that of sapinofuranone A and to have a structure represented by the following formula (I-4).

[Example 3] Activity test using cells Motor nerve (MN) is a major cell that forms a pyramidal tract that is a transmission path of voluntary movement, and is excited by glutamate input. In the pathological mechanism of ALS, conditions under which MN causes cell death include oxidative stress (reactive oxygen species ROS and active nitrogen derived from MN itself or surrounding cells during brain / spinal cord ischemia, neuroinflammation, etc. Species RNS causes oxidative damage, oxygen respiration damage, apoptosis, etc.), excitotoxicity (Ca2 ⁺ -dependent key protein associated with overloading of intracellular Ca ²⁺ caused by excessive glutamate stimulation, etc. TDP-43, etc.) And the like, and abnormal damage caused by accumulation of abnormal proteins (abnormality of cell metabolism based on the appearance and abnormal treatment of abnormal protein aggregates such as denatured SODl protein). Based on these hypotheses, for compound (I-1) (hereinafter sometimes referred to as “YY-1”), (1) inhibitory action of ROS generation due to serum shortage of medium, (2) under glutathione deficiency The inhibitory effect on excitotoxicity and (3) the inhibitory effect on cell death by temporarily expressed SODl-G93A were tested.

<Cells and media>
Mouse motor nerve cell line, NSC-34 cell (Neuroblastoma-Spinal Cord 34) was obtained from Dr. It was given by Neil Cashman via Keio University. Mouse motor neuroblastoma cells, N2a cells (Neuro 2a) were purchased from Sigma-Aldrich.

Tables 6 to 10 show the composition of the medium used in the test. Rich medium was used for NSC34 cell culture, and MEM medium was used for N2a cell culture.

<Applied instruments / apparatus / reagent>
The equipment, devices, and reagents used in the test are as follows.
・ Micro high-speed centrifuge CF16RX II (Hitachi)
・ CO ₂ incubator MCO-175 (SANYO)
・ Clean bench MCV-B131F (SANYO)
・ Fatal Bovine Serum (FBS) (HyClone SH30070)
・ Dulbecco's Modified Eagle's Medium (SIGMA D5796)
・ Minimum Essential Medium (MEM) (SIGMA M4655)
・ 100X Penicillin / Streptomycin (Invitrogen 15140, 10,000 U / mL penicillin and 10,000 mg / mL streptomycin included)

(ROS assay)
・ Fluorescent microplate reader BMG LABTECH FLUOStar OPTIMA (BMG LABTECH)
・ Hemocytometer Tiefe 0.100 mm 1/400 mm Thoma (NITIRIN)
・ DMEM ([-] phenol red) (GIBCO Invitrogen 31053-028)
・ CM-H ₂ DCFDA (5- (and-6) chlromethyl-2,7-dichlorodihydrofluoresceindiacetate, acetyl ester) (Invitrogen)
Trolox (CALBIOCHEM)
・ 96 well plate (IWAKI 3860-096)

(ODAP-EA assay)
・ DMEM (-Met / -Cys) (GIBCO Invitrogen 21013-024)
・ DMEM / F12 + GlutamaMAX-1 (Invitrogen 10565-018)
・ Horse serum (HS) (GIBCO Invitrogen 26050)
・ 200 mM L-glutamine (GIBCO 25030)
・ L-β-ODAP (β-N-oxalyl-L-α, β-diaminopropionic acid, manufactured by Lathyrus Technologies (Hyderabad500 007, India)
・ Ethacrynic Acid (Lot 105K1850) (SIGMA E4754-1G)
・ N-acetyl cysteine (NAC) (Lot 129K1944V) (SIGMA A7250-5G)
・ AlamarBlue (Lot 150844SA) (Nalgene)

(Dual-luciferase assay)
・ Opti-MEM (GIBCO 31985-070)
・ P3x plasmid (empty vector)
・ G93A plasmid (SOD1-G93A gene vector)
・ PGL4SV40 plasmid (firefly luciferase)
・ PGL4TK plasmid (Renilla luciferase)
・ Lipofectamine 2000 (Lot 1457325) (Invitrogen 11668-030)
・ N-2'-O-dibutyryladenosine 3 ', 5'-cyclic monophosphate sodium salt (db-cAMP) (SIGMA D0627)
・ Lactacystin (SIGMA L6785)
・ 5 × Passive Lysis Buffer (Promega E194A)
・ Stop & Glow Buffer (Promega E641A)
・ Stop & Glow Substrate (Promega E640A)
・ Luciferase Assay Buffer II (Promega E195A)
・ Luciferase Assay Substrate (Promega E151A)
・ Luminescencer-PSN (ATTO BIO-INSTRUMENT)
・ Luminometer tube (BD Falcon)
Shaker Titramax 100 (Heidolph)

<ROS assay>
The ROS assay is a test that evaluates the ability of cells to produce ROS under serum withdrawal stress and the test substance to eliminate the ROS. In the ROS assay, NSC-34 cells were cultured in a medium from which serum (FBS) had been removed, and the rise in ROS generated at that time was measured using CM-H ₂ DCFDA.

NSC-34 cells were suspended in phenol red free DMEM medium, and NSC-34 cells were seeded at a target concentration of 4 × 10 ⁴ cells / well in a 96-well plate at 200 μL / well. The well plate was incubated overnight in an incubator under conditions of 37 ° C. and 5% CO ₂ to allow the cells to settle.

Next, all the medium was removed from the 96-well plate, DMEM ([−] phenol red) was added at 200 μL / well, and then the test substance (or solvent alone) dissolved in DMSO was added at 1 μL / well. After incubating the well plate in an incubator at 37 ° C. under 5% CO ₂ for 3 hours, DMEM ([−] phenol red) was removed by 110 μL / well, and 5 μM CM-H ₂ DCFDA solution was added at 10 μL / well. Wells were added and placed in the incubator for 1 hour. After 1 hour, the fluorescence intensity (excitation wavelength: 485 nm, fluorescence wavelength: 520 nm) was measured using a fluorescence plate reader.

As a test substance for the ROS assay, a crude extract (322-Ext) obtained by extracting NUH322 strain culture solution after 3 weeks of culture with ethyl acetate and YY-1 were used. 322-Ext and YY-1 were used by dissolving in DMSO at a concentration of 10, 30 or 100 μg / μL, respectively. The test was performed 2 to 4 times in quadruplicate. As a positive control, 30 μg / mL (120 μM) of Trolox was used. From the measurement result obtained with the test substance, the relative intensity with respect to trolox was calculated by the following formula (1). In the following formula (1), “fluorescence intensity (sample)” represents the fluorescence intensity of the sample incubated with the test substance (322-Ext or YY-1 dissolved in DMSO), and “fluorescence intensity (solvent) “Represents the fluorescence intensity of the sample incubated with DMSO alone and“ fluorescence intensity (positive control) ”represents the fluorescence intensity of the sample incubated with 30 μg / mL trolox added.

The results are shown in FIG. Since YY-1 exhibited a concentration-dependent ROS scavenging (antioxidant) activity, it was confirmed that it has a ROS scavenging action of about 20% of trolox. YY-1 showed about 40% of the activity of 322-Ext at the same concentration.

<ODAP-EA assay>
The ODAP-EA assay reproduces a part of the cause of ALS, glutamate receptor-mediated excitotoxicity under mitochondrial oxidative stress, with L-β-ODAP and ethacrynic acid (EA), and the test substance is excitotoxic. It is a test to evaluate the effect of reducing The ODAP-EA assay was developed by the present inventors as a test for measuring the alleviating effect on motor neuronal excitotoxicity (Kusama-Eguchi K, et al., Food Chem Toxicol., 49, 636-643 (2011)). .). In the ODAP-EA assay, differentiated NSC-34 cells were added with ethacrylic acid (EA) having a mitochondrial glutathione level lowering effect, and L-β-ODAP, which is a neurotoxic amino acid, and added to NSC-34 cells. Excitotoxicity is produced and the test substance reduces the excitotoxicity.

NSC-34 cells were cultured in Rich medium in a 6 mL dish until confluent, and then the Rich medium in the dish was removed. Next, the Poor medium warmed to 37 ° C. was added to the dish and cultured in an incubator under 37 ° C. and 5% CO ₂ conditions. The Poor medium was replaced once a day for about 3 to 5 times to promote differentiation of NSC-34 cells.

NSC-34 cells differentiated as described above were suspended in Poor medium and seeded at a target concentration of 4 × 10 ⁴ cells / well in a 96-well plate at 200 μL / well. The well plate was incubated overnight in an incubator under conditions of 37 ° C. and 5% CO ₂ to allow the cells to settle.

Next, the Poor medium in the well plate was all removed, BM-E was added at 200 μL / well, and then the test substance was added at 1 μL / well and placed in an incubator for about 20 to 22 hours.

Next, 110 μL of the medium in the well plate was removed, AlamarBlue that had been brought back to room temperature in advance was added at 10 μL / well, and placed in an incubator under conditions of 37 ° C. and 5% CO ₂ . After about 1 hour, fluorescence intensity (excitation wavelength: 544 nm, fluorescence wavelength: 590 nm) was measured using a fluorescence plate reader.

As a test substance for the ODAP-EA assay, a crude extract (322-Ext) obtained by extracting the NUH322 strain culture solution after 3 weeks of culture with ethyl acetate and YY-1 were used. 322-Ext and YY-1 were used by dissolving in DMSO at the respective concentrations shown in FIG. In the test, 4 to 4 experiments of the same system were conducted 2 to 5 times. As a positive control, a final concentration of 4 mM NAC was used. From the measurement result obtained with the test substance, the relative intensity with respect to NAC was calculated by the following formula (2). In the following formula (2), “fluorescence intensity (sample)” represents the fluorescence intensity of the sample incubated with the test substance (322-Ext or YY-1 dissolved in DMSO), and “fluorescence intensity (solvent) "Represents the fluorescence intensity of the sample incubated with DMSO alone and" fluorescence intensity (positive control) "represents the fluorescence intensity of the sample incubated with 4 mM NAC added.

Results are shown in FIG. 322-Ext did not show ODAP-EA toxicity mitigation at most concentrations tested. On the other hand, YY-1 exhibited a strong ODAP-EA toxicity reducing effect at all concentrations of 0.1 to 100 μg / mL (0.55 to 550 μM). The relative intensity with respect to 4 mM NAC was highest when 0.1 to 3.0 μg / mL (0.55 to 16.5 μM) of YY-1 was added, and was about 20% or less. From these results, it was found that YY-1 is a major compound having ODAP-EA toxicity reducing activity among the compounds present in 322-Ext.

<Dual-Luciferase Assay>
SOD1-G93A is a causative gene of familial ALS and forms the background of proteinopathy. The Dual-luciferase assay is a test in which the SOD1-G93A gene is introduced into mouse motor neuroblastoma cells (N2a cells), and the test substance evaluates the effect of restoring the neurotoxicity caused by the temporary introduction of the gene into the cells. is there. In the Dual-luciferase assay, two types of luciferase plasmids are introduced as reporters into N2a cells together with SOD1-G93A plasmid, and the survival rate of the SOD1-G93A-introduced cells is evaluated by ATP-dependent bioluminescence.

(Seeding of N2a cells in a 24-well plate)
N2a cells were suspended in MEM medium and seeded in a 24-well plate at 500 × L / well at a target concentration of 10 × 10 ⁴ cells / well. The cells were then allowed to settle by placing in an incubator under conditions of 37 ° C. and 5% CO ₂ for 1 day.

(Transfection)
PGL4SV40 plasmid (firefly luciferase gene vector) and pGL4TK plasmid (Renilla luciferase gene vector) were added to P3x plasmid (empty vector) or G93A plasmid (SOD1-G93A gene vector), and a mixed plasmid solution was prepared using Opti-MEM. . A mixed solution of Opti-MEM ^™ and Lipofectamine 2000 was added to the mixed plasmid solution and left at room temperature for 5 minutes to prepare a plasmid solution for transfection.

All of the medium in the 24-well plate on which the N2a cells were fixed as described above was removed, and Opti-MEM previously warmed to 37 ° C. was added in an amount of 400 μL / well. Subsequently, 100 μL / well of the transfection plasmid solution was added. Thereafter, the plate was placed in an incubator under conditions of 37 ° C. and 5% CO ₂ for transfection. After 6 hours, the medium was removed from the plate, and 500 μL / well of MEM containing 2 μM dibutyryl-cAMP was added. Then, a test substance was added at 0.5 μL / well and incubated for 1 day in an incubator under conditions of 37 ° C. and 5% CO ₂ . Subsequently, 2 mM lactacystin (proteasome inhibitor) was added at 0.5 μL / well and further incubated for 1 day in an incubator under 37 ° C., 5% CO ₂ conditions.

(Measurement of bioluminescence)
On the third day, the medium was removed from the 24-well plate, 500 μL / well of 1 × PBS was added and removed several minutes later, and the inside of the well was washed. 1 × Passive Lysis Buffer was added at 100 μL / well, and shaken (215 rpm) for 15 minutes at room temperature to peel off the cells from the 24-well plate. The well was pipetted to collect the cell lysate, and the entire volume was transferred to a 1.5 mL microtube and centrifuged at 13,200 rpm at 4 ° C. for 1 minute.

Prepare a luminometer tube containing 50 μL of Luciferase Assay Reagent II (LAR II), add 10 μL of the supernatant after centrifugation to this, pipette 3 times, and then luminescence of firefly luciferase using the luminometer The strength was measured. Immediately after the measurement, 50 μL of Stop & Glow Reagent was added and vortexed lightly, and the luminescence intensity of Renilla luciferase was measured with a luminometer. Table 11 shows the composition of LAR II and Stop & Glow Reagent.

As test substances for the above-mentioned Dual-luciferase assay, a crude extract (322-Ext) obtained by extracting the NUH322 strain culture solution after 3 weeks of culture with ethyl acetate and YY-1 were used. 322-Ext and YY-1 were used by dissolving in DMSO at the respective concentrations shown in FIG. One or two tests were performed per system, 322-ext was performed twice, and YY-1 was performed 3-5 times. Resveratrol was used as a positive control. From the measurement result obtained with the test substance (sample), the relative intensity was calculated by the following formula (3). The relative intensity was similarly calculated for the positive control. In the following formula (2), “luminescence intensity (sample)” represents the luminescence intensity of a sample incubated by adding a test substance (322-Ext or YY-1 dissolved in DMSO). "Represents the fluorescence intensity of the sample incubated with DMSO alone.

The results are shown in FIG. YY-1 is about 40% higher than the luminescence intensity of the positive control at 0.1 μg / mL (0.55 μM) and 1 μg / mL (5.5 μM), and recovers SOD1-G93A genotoxicity comparable to 332-Ext Showed activity. From this result, it was found that YY-1 is a major compound having SOD1-G93A genotoxicity recovery activity among the compounds present in 322-Ext.

[Example 4] In vivo test using ALS model mice YY-1 had three kinds of activities in Example 3 above. Since NU was also a main metabolite of 322 cells and could be isolated in large quantities, experimental treatment using an ALS model animal was performed, and the in vivo activity was evaluated. All animal experiments were conducted as university-licensed animal experiments in accordance with the Science Council of Japan "Guidelines for Proper Implementation of Animal Experiments" and "Nihon University Animal Experiments Operational Regulations".

<Production of ALS model mouse>
(Mouse breeding environment)
The mice were bred under illumination at 8:00 to 20:00 under constant temperature and humidity conditions at 25 ° C. at the Laboratory Animal Center, Nihon University Pharmaceutical Research Institute. The experimental animals were raised by one animal per cage, and the flooring and cage were replaced once a week. Drinking water was used as a drop bottle, filtered water was used for animal drinking, and the contents were changed every two days. MF (feeding feed) was given as a bait, and a constant amount was always put in the bait and allowed to be freely ingested.

(Mating)
In the test, ALS model mice carrying the SOD1-G93A gene bred in-house were used. The first mouse was created by introducing a male mouse of B6SJL-Tg (SOD1-G93A) 1Gur / J, a 6-week-old SOD1-G93A transgenic mouse from Jackson Laboratory, USA to the mother at 8 weeks of age. Aged C57BL / 6J female mice were prepared and placed in the same cage for one week for mating.

After confirming the retention of the SOD1-G93A gene of the offspring mouse born by this mating by genotyping, it was crossed with C57BL / 6J female mice from about 6 weeks of age to produce the first backcross mouse (FuBk1). In addition, a female mouse to be multiplied with this FuBk1 male mouse was also produced at the same time. SJL / J male mice were crossed with C57BL / 6J female mice to produce offspring mice (F1).

Backcrossing was performed using the male mouse (FuBk1) carrying the first SOD1-G93A gene and the female mouse (F1) having a normal gene, which was produced as described above, and the resulting mouse was backcrossed 2 A male mouse that had retained the SOD1-G93A gene as a second mouse (FuBk2) was used in a preliminary test described later. A male mouse (FuBk2) carrying the SOD1-G93A gene was further crossed with an F1 female mouse to produce a third mouse (FuBk3) for backcrossing and a fourth mouse (FuBk4) for backcrossing, which will be described later. Used for this study.

(Genotyping)
Since SOD1-G93A gene is not inherited from all SOD1-G93A gene-bearing parent mice to all offspring mice, genotyping was performed on all born mouse mice to determine whether SOD1-G93A gene was retained. . Genotyping was performed by extracting DNA from the tail of the mouse, performing PCR using SOD1-G93A gene amplification primers, and confirming amplification of the SOD1-G93A gene fragment by agarose gel electrophoresis.

<Test substance evaluation method>
The test substance was evaluated based on the two points of survival days and motor function of mice. Regarding the survival days, the days until the mice were determined to be dead (including surrogate death) were evaluated. The motor function was evaluated by a motor test using the Rota-rod method.

(Determination of viability)
Mice that lost the specular reflex and failed to return to the correct posture within 20 seconds were determined to be surrogate death.

(Measurement of motor ability; Rota-rod test)
The Rota-rod test was performed using a ROTA-ROD TREADMILL for MICE MK-600 (Muromachi). In order to investigate the onset time of ALS-like symptoms in the test subject mice, the motor function of the mice was measured once a week by the Rota-rod method. The measurement method was to measure the number of seconds until the log could not be followed at 15 rpm (level 4) using a mouse that had been trained to walk three times or more in advance. Those who were able to continue walking for 300 seconds were determined to be undeveloped (exercise performance). In the case of falling, the measurement was performed again after resting for 10 minutes, and the exercise duration was measured again. Those that dropped twice in 300 seconds or less were considered as onset, and the number of seconds until they dropped was also measured.

<Statistical processing>
The test results were analyzed by the Kaplan-Meier method, and the p-value and median value were determined by the Log-Rank test or the Wilcoxon test. Movement disorders after onset were determined by T test for each point value.

<Administration test 1>
(Test animal)
Among the second back-mating mice (FuBk2) produced by crossing with normal females without gene mutations, male mice about 60 days old (presymptomatic stage) determined to retain the SOD1-G93A gene by genotyping A total of 16 animals were used as test animals.

(Test substance preparation and administration method)
The test substance was prepared at a concentration shown in Table 12 using a 2.5% DMSO solution as a solvent. The test substance was orally administered to mice using a sterilized disposable oral sonde. According to Table 13, the test substance was administered in an amount corresponding to the body weight. The administration group was allocated as shown in Table 12, and the test substance was orally administered at 100 μL / 10 g (body weight) once a day from 60 days after birth.

(Weight change)
FIG. 9 shows the change in the body weight of the mouse in administration test 1 during the test substance administration period. Body weight was measured twice a week. In FIG. 9, “YY-1 (1)” indicates a YY-1 (1 mg / kg) administration group, and “YY-1 (10)” indicates a YY-1 (10 mg / kg) administration group. Since no significant weight loss was confirmed in any of the YY-1 administration groups, the toxicity of YY-1 to the present ALS model mice was estimated to be low. Furthermore, since weight loss is thought to be based on muscle loss, YY-1 is likely to be effective in muscle loss in ALS patients.

<Dose test 2>
(Test animal)
FIG. 10A shows the results of analyzing the survival days of mice in the administration test 1 of Example 3 by the Kaplan-Meier method, and the analysis results of the YY-1 (10 mg / kg) administration group. FIG. 10B shows the results of analyzing the survival days of mice in the administration test 1 of Example 3 by the Kaplan-Meier method, and the analysis results of the YY-1 (1 mg / kg) administration group. A total of 51 male and female mice determined to have the SOD1-G93A gene by genotyping among the 3rd time of backcrossing mice (FuBk3) retaining the SOD1-G93A gene gene were used as test animals.

(Test substance preparation and administration method)
The test drug was prepared at a concentration shown in Table 14 using a 1% DMSO + 2% Tween 20 solution as a solvent. The test substance was orally administered to mice using a sterilized disposable oral sonde. According to Table 13 above, the test substance was administered in an amount corresponding to the body weight. The administration groups were allocated as shown in Table 14, and the test substance was orally administered to mice at 0.1 mL / 10 g (body weight) once a day from about 45 days after birth (5 mg / kg).

(Movement disorder)
FIG. 11A shows the results of analyzing the days until the onset of movement disorders in mice by the Kaplan-Meier method. In FIG. 11A, “YY-1” indicates a YY-1 (5 mg / kg) administration group. In the YY-1 (5 mg / kg) administration group, the number of days until the onset of motor dysfunction tended to be extended by about 6 days on average and 7 days on median compared to the control group (Control) (p). = 0.12).

(Survival days)
FIG. 11B shows the results of analyzing the survival days of mice by the Kaplan-Meier method. In the YY-1 (5 mg / kg) administration group, the survival days tended to be extended by about 5 days on average and 8 days on median for the control group (Control) (p = 0.090). .

<Administration test 3>
(Test animal)
Among the 4th backcross mice (FuBk4) produced by backcrossing the 3rd time backmale male mice that retain the SOD1-G93A gene gene, the SOD1-G93A gene is retained by genotyping. A total of 52 female mice determined were used as test animals.

(Test substance preparation and administration method)
The test drug was prepared at a concentration shown in Table 14 using 1% DMSO as a solvent. The test substance was administered to mice at a dose of 0.1 mL / 10 g (body weight) once a day from 105 days after birth using a sterilized disposable oral sonde (10 mg / kg).

(Movement disorder)
FIG. 12A shows the result of analyzing the course of movement disorders in mice. A significant difference was observed between the administration group and the control group in the period of 15.3 weeks to 18.3 weeks after birth, and a delay effect of about 7 days was observed in motor dysfunction.

(Survival days)
FIG. 12B shows the results of analyzing the survival days of mice by the Kaplan-Meier method. In the YY-1 administration group, the number of days of survival was significantly prolonged by about 6 days on average and 5 days on median compared to the control group (Control) (p = 0.029).

The above results indicate that YY-1 is a promising candidate for ALS treatment.

According to the present invention, there are provided a pharmaceutical composition for treating or preventing a neurodegenerative disease including ALS, and a novel compound used as an active ingredient of the pharmaceutical composition.

Claims (8)

1. A novel valerolactone compound represented by the following formula (I).
   

   

   [Wherein X is an allyl group, an aryl group, an ethynyl group, or a butenyl group. Y is a single bond or a hydroxymethylene group, and Z is an oxygen, methylene group or imide group. In the formula, Y is a single bond or a hydroxymethylene group. m is 0 or 1, and n is 1 or 2. ]
2. The compound represented by the formula (I) is the following (I-1):
   The novel compound of the valerolactone type according to claim 1.
3. A medicament comprising a compound represented by the formula (I) according to claim 1.
4. The pharmaceutical according to claim 3, comprising at least one of a stereoisomer, a pharmaceutically acceptable salt, and a pharmaceutically acceptable solvate as the compound represented by the formula (I). .
5. The medicament according to claim 3, comprising a pharmaceutically acceptable carrier.
6. The medicament according to claim 3, which treats or prevents neurodegenerative diseases or stroke.
7. The medicament according to claim 6, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).
8. The compound represented by the formula (I) is the following (I-1):
   

   

   The medicine according to claim 3.
   

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